Fluorescence detecting device with integrated circuit and photodiode, and detection method

ABSTRACT

A fluorescence detecting device is configured so that a semiconductor integrated circuit substrate includes a photodiode and a signal detecting circuit for detecting charges obtained as a result of photoelectric conversion by the photodiode, and a fluorescence reaction vessel where a fluorescence reaction occurs is arranged above the foregoing photodiode. Furthermore, in the device, an excitation-light-entry preventing layer is provided at one or more of a surface portion of the photodiode and a position between the photodiode and the fluorescence reaction vessel.

This application is a Division of application Ser. No. 10/154,095, nowU.S. Pat. No. 6,844,563 filed May 22, 2002, which patent is incorporatedherein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to a fluorescence detectingdevice for detecting a fluorescence reaction, for instance, afluorescence detecting device suitable for detection, etc., of aspecific gene contained in a sample.

2. Related Background Art

Recently, the genome sequence analysis has been developed significantly,and the determination of the whole base-sequence of the human genomewill be completed in 2003. Besides, the determination of genomes ofother creatures is proceeding throughout the world. With thisdevelopment of the genome analysis, the detection of genes has anincreased significance from the viewpoint of the determination offunctions of genes, the medical diagnosis, etc. Examples of conventionalgene detecting methods include the gene amplification methodsrepresented by the polymerase chain reaction (PCR) method, whilerecently the gene detecting method employing DNA chips is used widely.

A DNA chip is an approximately 1 cm×1 cm glass chip, silicon chip, etc.on which a plurality of single-strand DNAs are fixed. Examples of thesingle-strand DNAs to be fixed include DNAs as etiologic genes. The geneanalysis employing a DNA chip is performed, for instance, in thefollowing manner. First of all, a target gene is extracted from cells(for instance, blood cells). Then, the target gene is amplified by thePCR method. In the amplification, a fluorescent substance is employed tolabel an amplification product. A DNA chip is immersed in a solutioncontaining nucleic acid strands labeled with the fluorescent dye, sothat hybridization occurs. Thereafter, the DNA chip is washed so thatnucleic acids that have not been hybridized are removed.

Subsequently, the DNA chip is irradiated with an excitation light, andthe fluorescence is detected. An example of a fluorescence detectingdevice used herein is shown in FIG. 11. In the device, an excitationlight 309 from a light source 305 such as a laser is reflected by a beamsplitter 304, and enters an objective lens 306, where the light isfocused so as to be incident on a fixed portion 307 of a nucleic acidprobe on a DNA chip 308. In the case where a double strand is formed asa result of hybridization, a fluorescent substance is present on the DNAchip 308, and therefore, a fluorescence 310 is emitted upon theirradiation by the excitation light 309. Normally, the fluorescence 310and the excitation light 309 have a wavelength difference on the orderof several tens of nanometers. A part 311 of the fluorescence and areflected light of the excitation light 309 return to the objectivelens, and reach the beam splitter 304. Most of the reflected light ofthe excitation light 309 is reflected by the beam splitter 304, therebybeing directed to the light source side. The part 311 of thefluorescence passes through the beam splitter 204, thereby beingdirected to a photodetector 301. The part 311 of the fluorescence thathas passed through the beam splitter 304 passes through a filter 303that limits a wavelength, while the reflected light of the excitationlight 309 is blocked by the same. Furthermore, the part 311 of thefluorescence passes through a photodetector lens 302 and enters thephotodetector 301 for measuring an intensity of the fluorescence, wherethe fluorescence is detected.

However, the above-described conventional fluorescence detecting deviceis a large-scale and complex device having a long optical path, throughwhich the fluorescence is lost partly, thereby leading to a problem oflow detection sensitivity.

SUMMARY OF THE INVENTION

Therefore, with the foregoing in mind, it is an object of the presentinvention to provide a fluorescence detecting device that is small insize and has a high sensitivity.

To achieve the foregoing object, a first fluorescence detecting deviceof the present invention includes a semiconductor integrated circuitsubstrate including a photodiode and a signal detecting circuit fordetecting charges obtained as a result of photoelectric conversion bythe photodiode, and a fluorescence reaction vessel where a fluorescencereaction occurs, which is arranged above the photodiode. Here, thefluorescence reaction vessel may be displaced from a positionimmediately above the photodiode as long as at least a part of thefluorescence generated in the fluorescence reaction vessel enters thephotodiode. Normally, at least a part of the fluorescence reactionvessel is positioned above the photodiode. In the foregoing device, anexcitation-light-entry preventing layer is formed one or more of asurface portion of the photodiode and a position between the photodiodeand the fluorescence reaction vessel.

Thus, in the fluorescence detecting device of the present invention, thefluorescence reaction vessel is arranged above the photodiode thatdetects a fluorescence. This makes an optical path shorter in length,thereby ensuring the improvement of the fluorescence detectingsensitivity and the reduction of the overall size of the device.Furthermore, since this device includes an excitation-light-entrypreventing layer, the excitation light is prevented from entering thephotodiode. Therefore, it is possible to eliminate influences of theexcitation light. It should be noted that the excitation light entrypreventing layer is defined as a layer that prevents at least a part ofthe excitation light from entering the photodiode. Therefore, even if alayer slightly transmits the excitation light, it is acceptable as theexcitation light entry preventing layer.

The first device may be configured so that the photodiode includes ahigh-concentration first-conductivity-type semiconductor layer, asecond-conductivity-type semiconductor layer, and a low-concentrationfirst-conductivity-type semiconductor layer that are laminated in thestated order from a surface side of the photodiode, wherein, when areverse bias is applied, a part of the high-concentrationfirst-conductivity-type semiconductor layer is not depleted, and thesecond-conductivity-type semiconductor layer is depleted, and thehigh-concentration first-conductivity-type semiconductor layerconstitutes the excitation-light-entry preventing layer. The excitationlight has a wavelength shorter than that of a fluorescence and hence isconverted by the photoelectric conversion in a surface portion of thephotodiode. Therefore, the foregoing configuration prevents chargesgenerated from the excitation light in the vicinity of the surfaceportion from being accumulated in the photodiode.

Furthermore, in the first device, the excitation-light-entry preventinglayer may be a light absorbing layer that absorbs an excitation lightand is arranged between the photodiode and the fluorescence reactionvessel. Alternatively, the excitation-light-entry preventing layer maybe a light interference layer that reflects an excitation light arrangedbetween the photodiode and the fluorescence reaction vessel. Furtheralternatively, the excitation-light-entry preventing layer may be a gaslayer arranged between the photodiode and the fluorescence reactionvessel, and an excitation light is set so as to be incident in adirection such that the excitation light is reflected totally at aninterface between the gas layer and a bottom face of the fluorescencereaction vessel, where the refractive index decreases.

Furthermore, in the first device, a single-strand DNA may be fixed on aninternal bottom face of the fluorescence reaction vessel. In this case,it is used as a DNA chip. Alternatively, an antibody or an antigen maybe fixed on an internal bottom face of the fluorescence reaction vessel.Furthermore, in the foregoing fluorescence reaction vessel, a geneamplification reaction such as the PCR may be carried out so that anamplification product should be detected by fluorescence.

To achieve the aforementioned object, a second fluorescence detectingdevice of the present invention includes a semiconductor integratedcircuit substrate including a first photodiode, a second photodiode, anda signal detecting circuit for detecting electric signals from thephotodiodes, a fluorescence reaction vessel where a fluorescencereaction occurs, and a first optical filter and a second optical filterthat have different light transmission spectral characteristics fromeach other. In the device, the first optical filter is arranged abovethe first photodiode, the second optical filter is arranged above thesecond photodiode, and the fluorescence reaction vessel is formed abovethese optical filters, and the signal detecting circuit outputs afluorescence signal from which influences of an excitation light areeliminated. The fluorescence signal is derived based on a differencebetween the optical filters as to the ratio of the fluorescence signalto an excitation light signal, which difference depends on a differencebetween the light transmission spectral characteristics.

Thus, in the fluorescence detecting device of the present invention, thefluorescence reaction vessel is arranged above the photodiode thatdetects a fluorescence. This makes an optical path shorter in length,thereby ensuring the improvement of the fluorescence detectingsensitivity and the reduction of the overall size of the device.Furthermore, in the foregoing device, a fluorescence signal is derivedbased on a difference between the optical filters as to the ratio of thefluorescence signal to an excitation light signal, according to adifference between the light transmission spectral characteristics, withinfluences of an excitation light being eliminated therefrom. Therefore,it is possible to achieve the fluorescence detection with highprecision.

In the second device, one detection unit preferably is composed of thefirst photodiode and the first optical filter, plus the secondphotodiode and the second optical filter, and the device preferablyincludes a plurality of the detection units. This configuration allowsthe detection units to perform different tests, respectively.

Furthermore, in the second device, color filters having different colorsfrom each other preferably are used as these optical filters.

Furthermore, in the second device, a single-strand DNA may be fixed onan internal bottom face of the fluorescence reaction vessel. In thiscase, it is used as a DNA chip. Alternatively, an antibody or an antigenmay be fixed on an internal bottom face of the fluorescence reactionvessel. Furthermore, in the foregoing fluorescence reaction vessel, agene amplification reaction such as the PCR may be carried out so thatan amplification product should be detected by fluorescence.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a cross-sectional view illustrating an example of a firstfluorescence detecting device of the present invention.

FIG. 2A is a circuit diagram of the device shown in FIG. 1, and FIG. 2Bis a driving timing chart of the same.

FIG. 3 is a perspective view of the device shown in FIG. 1.

FIG. 4 is a cross-sectional view illustrating another example of thefirst fluorescence detecting device of the present invention.

FIG. 5 is a cross-sectional view illustrating still another example ofthe first fluorescence detecting device of the present invention.

FIG. 6 is a circuit diagram illustrating still another example of thefirst fluorescence detecting device of the present invention.

FIG. 7 is a cross-sectional view of the device shown in FIG. 6, which istaken along a line I—I shown therein.

FIG. 8 is a cross-sectional view illustrating an example of a secondfluorescence detecting device of the present invention.

FIG. 9A is a graph illustrating spectral characteristics of an absorbedlight and a fluorescence of a fluorescent material, and FIG. 9B is agraph illustrating light transmission spectral characteristics ofoptical filters.

FIG. 10 is a perspective view of the device shown in FIG. 8.

FIG. 11 is a view illustrating a configuration of a conventionalfluorescence detecting device.

DETAILED DESCRIPTION OF THE INVENTION

First Embodiment

FIGS. 1, 2, and 3 illustrate an example of a basic configuration of afluorescence detecting device of the present invention. FIG. 1 is across-sectional view illustrating a structure of a photodetector part ofthe foregoing device, and FIG. 3 is a perspective view of thephotodetector part of the foregoing device. FIG. 2A is a circuit diagramof a fluorescence detecting circuit of the foregoing device, and FIG. 2Bis a driving timing chart of the fluorescence detecting circuit. InFIGS. 1 and 3, the same elements are designated by the same referencenumerals.

As shown in FIG. 3, the photodetector part of the foregoing deviceincludes a semiconductor integrated circuit substrate 15 and afluorescence reaction vessel composed of a transparent container 14, asprincipal constituent elements. The fluorescence reaction vesselcontains a fluorescence reaction solution 17. On the semiconductorintegrated circuit substrate 15, a fluorescence detecting circuit isprovided. It should be noted that 16 denotes an excitation lightentering the fluorescence reaction vessel.

As shown in FIG. 2A, the fluorescence detecting circuit includes aphotodiode 22, an amplifying transistor 24 whose gate is fed with avoltage of the photodiode 22, a reset transistor 23 that resets chargesof the photodiode 22, and a load transistor 25. In the fluorescencedetecting circuit, the amplifying transistor 23 and the load transistor25 constitute a source follower circuit. It should be noted that, inFIG. 2A, 32 denotes a power source of the amplifying transistor 24, 33denotes a reset power source of the reset transistor 23, and 34 denotesa timing control input terminal of the reset transistor. 35 denotes asignal output terminal of the source follower circuit composed of theamplifying transistor 24 and the load transistor 25.

The fluorescence detecting circuit operates in the following manner.First of all, a cathode side of the photodiode 22 is charged by thereset transistor 23 so as to have a positive voltage. Here, the sourcefollower circuit composed of the amplifying transistor 24 and the loadtransistor 25 has a signal output substantially equal to a gate voltageof the amplifying transistor 24. Since the gate of the amplifyingtransistor 24 is connected with the cathode of the photodiode 22, thesignal output terminal 35 has a voltage substantially equal to a voltageof the reset power source 33 upon resetting. In this state, when lightis incident on the photodiode 22, electrons are generated byphotoelectric conversion. The electrons are accumulated in a n-typeimpurity layer 8 forming the photodiode 22, thereby lowering the voltageon the cathode side of the photodiode 22. This causes the gate voltageof the amplifying transistor 24 to decrease, thereby causing the voltageof the signal output terminal 35 to decrease also. This sequence ofoperations can be illustrated by a timing chart as shown in FIG. 2B.More specifically, when a transition to a high level is made at thetiming control input terminal 34 of the reset transistor 23 (thetransistor is turned on), the signal output terminal 35 is charged so asto have a voltage of the reset power source 33. Thereafter, when atransition to a low level is made at the timing control input terminal34 of the reset transistor 23 (the transistor is turned off), thephotoelectric conversion by the photodiode 22 causes a change in thevoltage at the timing control input terminal 34 of the reset transistor23, and likewise causes a change in the voltage of the signal outputterminal 35. The voltage of the signal output terminal 35 indicates anintensity of light subjected to the photoelectric conversion.

FIG. 1 is a cross-sectional view of a photodetector part of theforegoing device. A photodiode, an amplifying transistor, and a resettransistor are formed on this part of the semiconductor integratedcircuit substrate 15. As shown in the drawing, the foregoing photodiodeis configured so that an n-type impurity layer 8(second-conductivity-type semiconductor layer) is formed on a surfacepart of a p-type silicon substrate 7 (low-concentrationfirst-conductivity-type semiconductor layer), and further, a p+-typeimpurity layer 21 (high-concentration first-conductivity-typesemiconductor layer) is formed in a surface portion thereof. The p+impurity layer 21 composing the photodiode is connected to a constantvoltage source. Moreover, the photodiode is connected electrically withthe gate 3 of the amplifying transistor 6 via a metal wire 1. 2 and 5denote a source metal wire and a drain metal wire of the amplifyingtransistor 6, respectively. The foregoing photodiode is connectedelectrically with the reset transistor 9. 11 denotes a gate of the resettransistor 9, and 10 denotes a reset power source line. 12 denotes ametal layer that shields elements other than the photodiode (forinstance, active elements such as the reset transistor). 4 denotes aninterlayer insulation layer. The fluorescence reaction vessel 13 isarranged above the foregoing photodiode.

In the foregoing device, when an excitation light 16 is applied to thefluorescence reaction vessel 13, a part of a fluorescence generated inthe fluorescence reaction vessel 13 is incident on the photodiode, wherethe light is subjected to the photoelectric conversion, whereby signalcharges are generated. The signal charges are accumulated in the n-typeimpurity layer 8, and a voltage according to the foregoing charges isfed to the gate of the amplifying transistor 6. Here, it should be notedthat any charges remaining in the photodiode are discharged by the resettransistor before the signal charges are accumulated. Thereafter thephotodetector circuit operates as described above, whereby lightsubjected to the photoelectric conversion is detected.

In the foregoing device, the p+ impurity layer 21 composing thephotodiode functions as an excitation-light-entry preventing layer thatprevents photodiode are discharged by the reset transistor before thesignal charges are accumulated. Thereafter the photodetector circuitoperates as described above, whereby light subjected to thephotoelectric conversion is detected.

In the foregoing device, the p+ impurity layer 21 composing thephotodiode functions as an excitation-light-entry preventing layer thatprevents or suppresses the entry of charges generated by thephotoelectric conversion of the excitation light into the n-typeimpurity layer 8. When light enters a material having anoptical-absorption coefficient α, a charge generation ratio g(d)indicative of a ratio of charges obtained by photoelectric conversion ata depth of d is expressed as γ·Φ·α·exp(−α·d), where γ represents aquantum efficiency, and Φ represents a flux density. Since theoptical-absorption coefficient a of silicon increases as the wavelengthdecreases in the vicinity of the visible light range, light with a shortwavelength tends to be subjected to the photoelectric conversion andabsorbed in the vicinity of a surface of the layer. When the excitationlight applied thereto for exciting a fluorescence has a wavelengthshorter than that of the fluorescence, the excitation light is absorbedin a region closer to the surface than the region where the fluorescenceis absorbed. Thus, charges generated by the excitation light areabsorbed by the p+ impurity layer 21, thereby resulting in that chargesoriginating from the excitation light, which are unnecessary, hardly areaccumulated in the n-type impurity layer 8.

In the foregoing device, the semiconductor integrated circuit substrate15 is produced by, for instance, the MOS (metal-oxidefilm-semiconductor) process for producing an integrated circuit (IC)with a silicon substrate 7, but it is not limited to this in the presentinvention. The semiconductor integrated circuit substrate 15 may be, forinstance, a polycrystalline silicon integrated circuit substrate, anamorphous silicon integrated circuit substrate, or a GaAs integratedcircuit substrate formed on a glass substrate. Furthermore, thetransparent container 14 composing the fluorescence reaction vessel 13can be made of, for instance, quartz, polymethyl methacrylate (PMMA),etc., but the material is not limited to these. Any material may be usedas long as it has a high light transmittance and emits the leastpossible fluorescence. Furthermore, in the foregoing device, theinterlayer insulation film 4, that is, the surface part of thesemiconductor integrated circuit substrate 15, preferably is flattenedby the chemical machining process (CMP) or the like. In the case wherethe semiconductor integrated circuit substrate 15 has a flat surface, itis foregoing device is performed, for instance, in the following manner.First of all, a single-strand DNA with the complementary sequence tothat of a gene as a target of the detection is fixed in the fluorescencereaction vessel. The fixing method is not limited particularly, and anyusual method may be used. A DNA (oligonucleotide) may be synthesizeddirectly on a bottom of the fluorescence reaction vessel, oralternatively, the bottom of the fluorescence reaction vessel may becoated with a material to which a DNA tends to be bound, and a clonedDNA or a PCR product may be fixed thereon. Then, a sample solution isintroduced into the fluorescence reaction vessel. Here, in the casewhere the target DNA itself is labeled with a fluorescent dye such asCy3, the fluorescence reaction vessel may be washed after the samplesolution is introduced. Even in the case where the target DNA is notlabeled with a fluorescent dye, a fluorescent intercalator such asSYBR-Green or the like may be put in the sample solution or thefluorescence reaction vessel. Then, the excitation light is projectedinto the fluorescence reaction vessel, for example, from a side thereof.In the case where the single-strand DNA fixed on the bottom of thefluorescence reaction vessel and the target DNA are hybridized therebyforming a double strand, a fluorescence is emitted radially by eitherthe fluorescent intercalator or the fluorescent label of the target DNA,which has entered in the double strand. In the case where the SYBR-Greenis used, a second harmonic generation (SHG) laser with a wavelength of473 nm may be projected thereto as the excitation light. A part of thefluorescence emitted is detected by the photodiode, and converted by thephotoelectric conversion into electric signals. Thereafter, theaforementioned operations are carried out by the semiconductorintegrated circuit substrate, whereby electric signals according to theforegoing fluorescence are output.

In the case where the foregoing device is configured so as to include aplurality of photodiodes and single-strand DNAs of a plurality of typesare fixed accordingly, a plurality of samples can be analyzed in onedetecting operation.

Furthermore, though a single-strand DNA is fixed on the bottom of thefluorescence reaction vessel in the foregoing case described as anexample, an antibody or an antigen may be fixed instead. In this case, asample solution of a fluorescent-labeled antibody or antigen is put inthe fluorescence vessel. Thereafter, the sample solution is removed, andthe excitation light for fluorescence is projected thereto. Here, in thecase where an antigen-antibody complex is formed, a fluorescence isemitted, which is detected by a photodiode. Alternatively, the enzymeimmunoassay (ELISA) may be applied. In this case, a first antibody isfixed on the bottom of the fluorescence reaction vessel, to which asample solution containing an antigen is supplied. Then, anantigen-antibody complex is formed. Further, a second antibody, which isenzyme-labeled, is supplied thereto, so that a complex is formed in asandwich structure in which the first antibody, the antigen, the secondantibody are arranged in the stated order. Then, a substrate that ischanged to a fluorescent substance by an enzyme reaction is addedthereto, so as to be subjected to an enzyme reaction. The excitationlight is projected thereto, and a fluorescence of the fluorescentsubstance produced is detected by the photodiode. It should be notedthat in the fluorescence detection by the antibody-antigen reaction, inthe case where a plurality of photodiodes are provided, a plurality ofantibodies or antigens may be fixed so that they can be analyzed atonce.

Furthermore, in this device, the gene amplification such as the PCR maybe performed. In this case, a sample solution containing a target DNA,and a buffer solution containing a pair of primers that can behybridized with both ends of the target DNA, a heat-resistant DNApolymerase (TaqDNA polymerase, etc.), dNTPs, a fluorescent intercalator,and the like are put in the foregoing fluorescence reaction vessel.Then, by repeating a series of steps of the denaturation of the targetDNA with heat, the annealing of the primers, and the elongation of theDNA polymerase, the target DNA is amplified. Since the fluorescentintercalator is bound to an amplification product obtained, irradiationwith the excitation light causes a fluorescence to be emitted, which canbe detected by the photodiode.

Second Embodiment

FIG. 4 is a cross-sectional view illustrating another example of thefluorescence detecting device of the present invention. In the drawing,the same elements as those in FIG. 1 are designated by the samereference numerals.

The device includes a filter layer 18 that absorbs or reflects anexcitation light. The filter layer 18 is provided between the n-typeimpurity layer 8 and a bottom of the fluorescence reaction vessel 13. Asa material of the filter layer 18, any material may be used as long asit transmits a fluorescence but hardly transmits the excitation light16. For instance, a pigment film, a multilayer interference film, a dyedfilm, a colored glass, etc. may be used. For instance, in the case wherethe excitation light has a wavelength of 497 nm and the fluorescence hasa wavelength of 520 nm, a pigment film that absorbs light with awavelength of not more than 510 nm may be used as the filter layer 18.In the case where a multilayer interference film is used as the filterlayer 18, the filter layer can be formed by producing a semiconductorintegrated circuit substrate and laminating materials having differentrefractive indices from each other on the surface of the board so as toform a multilayer film. This multilayer film can be formed by laminatingsilicon dioxide and titanium oxide, or alternatively, silicon dioxideand silicon nitride, by sputtering or chemical vapor deposition (CVD).

Furthermore, by measuring the light transmission spectralcharacteristics of the filter layer 18 and the spectral characteristicsof the fluorescence and the excitation light beforehand, it is possibleto calculate a ratio between the transmittance of the filter layer withrespect to the fluorescence and the transmittance of the filter layerwith respect to the excitation light. With such a transmittance ratiodetermined, it is possible to calculate a more accurate fluorescenceintensity based on the value (light intensity) measured by the foregoingdevice and the foregoing ratio.

The other configurations, conditions, operations, etc. of this deviceare identical to those in the first embodiment described above.

Third Embodiment

FIG. 5 is a cross-sectional view illustrating still another example ofthe fluorescence detecting device of the present invention. In thedrawing, the same elements as those in FIG. 1 are designated by the samereference numerals.

This device is configured so as to include a transparent layer 20 madeof quartz glass on a bottom face of the fluorescence reaction vessel 13,and a space between the transparent layer 20 and the n-type impuritylayer 8. In this space between the transparent layer 20 and the n-typeimpurity layer 8, a gas, for instance, air, is present, thereby forminga gas layer 19. Since the air has a refractive index of 1.0 and thequartz glass has a refractive index of 1.5, the refractive indexdecreases at an interface between the transparent layer 20 and the gaslayer 19, thereby causing light entering from the quartz glass layer 20to the gas layer 19 to be reflected totally, provided that the light hasan incident angle of not less than approximately 44° with respect to anormal direction. Therefore, in the case where the excitation light 16is caused to be incident thereon, for instance, with an incident angleof 60° with respect to the normal direction, the excitation light isreflected at the interface between the transparent layer 20 and the gaslayer 19, thereby not entering the n-type impurity layer 8. Therefore,it is possible to cause only the fluorescence to enter the n-typeimpurity layer 8.

The gas layer 19 is not limited to the air layer, but any may be used aslong as it has a suitably small refractive index. For instance, the gaslayer may be a nitrogen layer, or a vacuum layer. Besides, the materialforming the transparent layer 20 is not limited to quartz glass, but anytransparent material that transmits a fluorescence and has a refractiveindex higher than that of the gas layer 19 may be used. For instance,polymethyl methacrylate, non-fluorescent glass, etc. may be used.

The other configurations, conditions, operations, etc. of the foregoingdevice are identical to those in the first embodiment described above.

Fourth Embodiment

FIG. 6 is a plan view illustrating still another example of thefluorescence detecting device of the present invention. FIG. 7 is across-sectional view of the device taken in a line I—I direction shownin FIG. 6. In these drawings, the same elements are designated by thesame reference numerals.

As shown in FIG. 6, a photodetector part of the foregoing deviceincludes a semiconductor integrated circuit substrate 147 and atransparent container 135 composing a fluorescence reaction vessel 140made of a transparent material as principal constituent elements. Theforegoing fluorescence reaction vessel 140 contains a fluorescencereaction solution. On the foregoing semiconductor integrated circuitsubstrate 147, there are formed a plurality of photodiodes 102 arrayedtwo-dimensionally, a Y transfer section 101, an X transfer section 103,a charge accumulating section 104, and an amplifying circuit 115. The Ytransfer section 101 and the X transfer section 103 are so-called chargecoupled devices (CCD), each of which has a plurality of transferelectrodes arrayed in a transfer direction. The amplifying circuit 115is a signal detecting circuit for detecting charges accumulated in thecharge accumulating section 104, and includes an amplifying transistor106 whose gate is fed with a voltage of the charge accumulating section104, a reset transistor 107 that resets charges of the chargeaccumulating section 104, and a load transistor 105. In the amplifyingcircuit 115, the amplifying transistor 106 and the load transistor 105constitute a source follower circuit. It should be noted that in FIG. 6,108 denotes a gate of the amplifying transistor 106, 114 denotes a resetpower source, 113 denotes a reset pulse (r) terminal, 109 denotes a gatepower source of the load transistor, 112 denotes a power source of thesource follower circuit, 110 denotes a ground power source, and 111denotes a signal output terminal.

When an excitation light 141 is applied to the fluorescence reactionvessel 140 a fluorescence is generated, and when the fluorescence entersthe photodiodes 102, it is subjected to the photoelectric conversion,whereby charges are accumulated therein. The charges thus obtained bythe photoelectric conversion and accumulated are moved to the Y transfersection 101 by a readout operation 121. The charges moved to the Ytransfer section 101 are transferred to the X transfer section 103 by atransfer operation 122 of applying a pulse voltage to a plurality oftransfer electrodes of the Y transfer section 101. Then, the chargestransferred to the X transfer section 103 are transferred to the chargeaccumulating section 104 by a transfer operation 123 of applying a pulsevoltage to a plurality of transfer electrodes of the X transfer section103. Through these operations, the charges obtained by the photoelectricconversion by the photodiodes 102 are accumulated in the chargeaccumulating section 104.

The following will describe an operation performed by the amplifyingcircuit 115. Before charges are accumulated in the charge accumulatingsection 104, a pulse that turns on the reset transistor 107 is fed fromthe reset pulse terminal 113 to a gate of the reset transistor 107, sothat the charge accumulating section 104 is charged to have a voltage ofthe reset power source 114. This reset operation causes the chargeaccumulating section 104 to have a voltage of the reset power source114. Thereafter, the charges generated by the photoelectric conversionof the fluorescence are accumulated in the charge accumulating section104, thereby causing a change in the voltage of the charge accumulatingsection 104. The charge accumulating section 104 is connected with thegate 118 of the amplifying transistor 106, and hence, the gate 118 has avoltage equal to that of the charge accumulating section 104. Since theamplifying transistor 106 and the load transistor 105 composes a sourcefollower circuit, the signal output terminal 111 (Vo) has a voltage thatis substantially equal to the voltage of the gate 118. With this voltageof the signal output terminal 111 (Vo), an intensity of the fluorescencecan be determined. In other words, when the fluorescence is intense,charges obtained by the photoelectric conversion increase, therebylowering the voltage of the charge accumulating section 104.Consequently, the voltage of the signal output terminal 111 (Vo) drops.When the fluorescence is weak, charges Since the amplifying transistor106 and the load transistor 105 composes a source follower circuit, thesignal output terminal 111 (Vo) has a voltage that is substantiallyequal to the voltage of the gate 118. With this voltage of the signaloutput terminal 111 (Vo), an intensity of the fluorescence can bedetermined. In other words, when the fluorescence is intense, chargesobtained by the photoelectric conversion increase, thereby lowering thevoltage of the charge accumulating section 104. Consequently, thevoltage of the signal output terminal 111 (Vo) drops. When thefluorescence is weak, charges obtained by the photoelectric conversiondecrease, thereby causing the voltage of the signal output terminal 111(Vo) to approximate a voltage of the reset power source 114, which is ahigh voltage.

As shown in FIG. 7, in the foregoing device, a photodiode is composed ofa p-type semiconductor substrate 131 (low-concentrationfirst-conductivity-type semiconductor layer), a n-type impurity layer132 (second-conductivity-type semiconductor layer), and a p+ impuritylayer 133 (high-concentration first-conductive-type semiconductorlayer), so that the same mechanism as that in the first embodimenteliminates influences of the excitation light.

Furthermore, the foregoing device includes readout transistors 116 forreading out charges generated by the photoelectric conversion, n-typeimpurity layers 134 that serve as channels of the Y transfer section101, an interlayer insulation film 138, polysilicon layers 137,shielding metal layers 137, and a transparent container 135 composing afluorescence reaction vessel 140. The polysilicon layers 137 serve astransfer electrodes of the Y transfer section 101, as well as gates ofthe readout transistor 116. The fluorescence from the fluorescencereaction solution 139 is subjected to the photoelectric conversion atthe photodiode, and signal charges generated therein are accumulated inthe n-type impurity layer 132 of each photodiode. It should be notedthat before accumulating the signal charges, the n-type impurity layers132 are depleted. Then, a high voltage is applied to the polysiliconlayers 137 so as to move the charges accumulated in the n-type impuritylayers 132 to the n-type impurity layers 134 of the Y transfer section101. This operation is equivalent to the readout operation 121 shown inFIG. 6.

Thus, an effect of the suppression of influences of charges originatingfrom the excitation light can be achieved also by transferring signalscharges from the photodiode to an accumulation capacitor such as acharge coupled device (CCD) temporarily, and thereafter feeding the sameto the amplifying circuit (signal detecting circuit). detecting deviceof the present invention. FIG. 8 is a cross-sectional view illustratinga structure of a photodetector portion of the foregoing device, and FIG.10 is a plan view illustrating the photodetector portion of the device.In the foregoing drawings, the same elements are designated by the samereference numerals.

As shown in the drawings, the photodetector portion of the deviceincludes a semiconductor integrated circuit substrate, first and secondoptical filters 208 and 209 that have different light transmissionspectral characteristics from each other, and a fluorescence reactionvessel 206 made of a transparent material, as principal constituentelements. The fluorescence reaction vessel 206 contains a fluorescencereaction solution 207 containing a fluorescent material. Furthermore, afluorescence detecting circuit is formed on the semiconductor integratedcircuit substrate. It should be noted that 213 denotes an excitationlight incident on the fluorescence reaction vessel 206.

As shown in FIG. 10, the fluorescence detecting circuit includes twophotodiodes 202-1 and 202-2, an amplifying circuit 211 to which signalsfrom the photodiodes are supplied, and reset transistors 210 that resetsignals of the photodiodes (put the photodiodes in a reversely biasedstate). The amplifying circuit 211 performs a specific calculation withsignals supplied from the photodiodes, and outputs, as a result of thecalculation, a signal according to a fluorescence, from which influencesof the excitation light have been eliminated. The calculation will bedescribed later. It should be noted that in FIG. 10, 212 denotes asignal output terminal of the amplifying circuit 211, 214 denotes areset pulse terminal, and 215 denotes a reset power source of the resettransistors 210.

FIG. 8 is a cross-sectional view of the photodetector portion of theforegoing device. Two n-type impurity layers 202-1 and 202-2 are formedon a p-type silicon substrate 201, and a high-concentration p-typeimpurity layer 203 is formed thereon, whereby two photodiodes (first andsecond photodiodes) are formed. On the two photodiodes, a green-colorfilter 208 (first optical filter) and a blue-color filter 209 (secondoptical filter) are arranged, respectively, with an interlayer film anda passivation film 204 interposed therebetween. Furthermore, afluorescence reaction vessel 206 is formed thereon, with a transparentflattening film 205 interposed therebetween. The foregoing filters arecolor filters containing pigments. Furthermore, as described above, thefluorescence reaction vessel 206 contains the fluorescence reactionsolution 207. In this device, the p-type silicon substrate 201 and thehigh-concentration p-type impurity layer 203 are grounded. While thedevice operates, the photodiodes are put in a reversely biased statebefore a fluorescence is subjected to the photoelectric conversion aswill be described later. Accordingly, the impurity concentration of thehigh-concentration p-type impurity layer 203 is set so that the layer isnot depleted completely even when the photodiodes are put in thereversely biased state.

In the device according to the present embodiment also, as in the firstembodiment, the semiconductor integrated circuit substrate can be madeof, for instance, a silicon substrate, but it is not limited to this inthe present invention. The semiconductor integrated circuit substratemay be, for instance, a polycrystalline silicon integrated circuitsubstrate, an amorphous silicon integrated circuit substrate, or a GaAsintegrated circuit substrate formed on a glass substrate. In this devicealso, the semiconductor integrated circuit substrate preferably has aflattened surface. Besides, the fluorescence reaction vessel can beformed by arranging a transparent container on a semiconductorintegrated circuit substrate, as shown in the drawing. The transparentcontainer may be made of, for instance, quartz, or polymethylmethacrylate (PMMA), but the material is not limited to these. Anymaterial may be used as long as it has a high light transmittance andemits the least possible fluorescence.

The following will describe an operation of the foregoing device. Firstof all, a pulse is supplied to the reset pulse terminal 214, to turn onthe reset transistors 210, thereby charging the n-type impurity layers202-1 and 202-2 of the photodiodes so that they have positive voltages.This operation puts the photodiodes in the reversely biased state.

Then, the reset transistors 210 are turned off, and detection of afluorescence is carried out. When an excitation light 213 is applied tothe fluorescence reaction vessel 206, a part of a fluorescence generatedin the fluorescence reaction vessel 206 is incident on the photodiodes,where the light is subjected to the photoelectric conversion, wherebysignal charges are generated. The signal charges are accumulated in then-type impurity layers 202-1 and 202-2, and electric signals accordingto the foregoing charges are fed to the amplifying transistor 211. Here,a signal from the photodiode 213-1 and a signal from the photodiode213-2 are supplied to the amplifying circuit 211, and a fluorescenceintensity is calculated based on these signals.

The detection of a fluorescence will be described in more detail below,by taking as an example a case where a light with an absorption peakwavelength of 497 nm, which provides the highest fluorescenceefficiency, is employed as an excitation light, and SYBR-Green I(intercalator) is employed as a fluorescent. FIG. 9A shows a lightabsorption spectral characteristic 221 and a fluorescence spectralcharacteristic 222 of SYBR-Green I (fluorescent). 223 indicates aposition of the excitation light wavelength of 497 nm in the graph. Asshown in the drawing, the fluorescence has an emission peak on a longwavelength side as compared with a light absorption peak thereof. On thephotodiodes, the excitation light and the fluorescence are incidentafter having passed through the green-color filter 208 or the blue-colorfilter 209. FIG. 9B shows a transmission spectral characteristic 225 ofthe green-color filter and a transmission spectral characteristic 224 ofthe blue-color filter. The difference between the excitation light andthe fluorescence in the spectral characteristics as shown in FIG. 9A andthe difference between the filters in the spectral characteristics asshown in FIG. 9B make the signal from the n-type impurity layer 202-1under the green-color filter 208 and the signal from the n-type impuritylayer 202-2 under the blue-color filter 209 different from each other.Since the green-color filter 208 has a light transmission peak close tothe peak wavelength of the fluorescence, the photodiode including then-type impurity layer 202-1 performs more efficient photoelectricconversion with respect to the fluorescence than with respect to theexcitation light. In contrast, since the blue-color filter 209 has alight transmission peak far from the peak wavelength of thefluorescence, the photodiode including the n-type impurity layer 202-2performs more efficient photoelectric conversion with respect to theexcitation light than with respect to fluorescence. A ratio between alight transmittance with respect to the excitation light and a lighttransmittance with respect to the fluorescence can be determinedbeforehand as to each of the green-color filter 208 and the blue-colorfilter 209, based on the spectral characteristics of the excitationlight and the fluorescence. According to the ratios of lighttransmittances thereof and the signals from the n-type impurity layers202-1 and 202-2, a fluorescence intensity can be calculated. In thiscase, since the green-color filter 208 efficiently transmits thefluorescence, the measurement with a high-sensitivity can be achieved.

One example of the foregoing calculation is shown, with reference toFIGS. 9A and 9B. For instance, it is assumed that in the case where afluorescence is obtained with an excitation with a wavelength of notmore than 497 nm, a light having passed through a filter with thespectral characteristic 225 has an intensity of I1 and includes anexcitation light component and a fluorescence component at a ratio of1:2, and a light having passed through a filter with the spectralcharacteristic 224 has an intensity of I2 and includes an excitationlight component and a fluorescence component at a ratio of 2:1. In thiscase, the fluorescence intensity can be determined by the followingarithmetic expression:Fluorescence Intensity=⅔(I 1−I 2/2)To determine an intensity of the fluorescence with an excellentprecision with the foregoing calculation, it is desirable that thefluorescence content is small in the light having passed through thefilter with the spectral characteristic 224.

It should be noted the filters do not necessarily have the spectralcharacteristics as shown in FIG. 9B, and two or more types of filtersthat differ in the transmission peak wavelength and the distributionform will suffice. For instance, a peak wavelength of transmission lightpassing through one of the filters is set close to a peak wavelength ofa fluorescence so that the filter efficiently transmits thefluorescence, and does not transmit much of the excitation light. Here,the other filter is made to have a distribution characteristic so as toinclude a larger proportion of the excitation light component. Thedifference between the peak wavelengths of the two filters preferably isgreater than a difference between those of the excitation light and thefluorescence. Besides, a half width of the distribution of each of thefilters preferably is small; more preferably, it is smaller than adifference between the wavelengths of the excitation light and thefluorescence. The filters having different light transmission spectralcharacteristics are not limited to the pigment color filters. Forinstance, interference filters, dyed filters, or colored glass can beused as the foregoing filters.

It should be noted that an operation of the foregoing device fordetecting a fluorescence of a gene can be performed in the same manneras that in the first embodiment.

Furthermore, as described above, it is preferable that a plurality ofdetection units are provided, each of which is composed of the firstphotodiode and the first optical filter, plus the second photodiode andthe second optical filter. This configuration allows the detection unitsto perform different tests, respectively. For instance, by providing aplurality of the detection units and fixing a plurality of types ofsingle-strand DNAs in the detection units accordingly, a plurality oftests can be carried out with one fluorescence detection.

The invention may be embodied in other forms without departing from thespirit or essential characteristics thereof The embodiments disclosed inthis application are to be considered in all respects as illustrativeand not limiting. The scope of the invention is indicated by theappended claims rather than by the foregoing description, and allchanges which come within the meaning and range of equivalency of theclaims are intended to be embraced therein.

1. A fluorescence detecting device comprising: a semiconductorintegrated circuit substrate including a plurality of photodiodes and asignal detecting circuit for detecting charges obtained as a result ofphotoelectric conversion by the plurality of photodiodes; and afluorescence reaction vessel where a fluorescence reaction occurs, thefluorescence reaction vessel being formed on the plurality of thephotodiodes, with a plurality of photodiodes provided per onefluorescence reaction vessel; wherein the fluorescence reaction in theone fluorescence reaction vessel generates fluorescence that enters theplurality of photodiodes and is subjected to the photoelectricconversion, and wherein an excitation-light-entry preventing layer isformed at either or both of a surface portion of the plurality ofphotodiodes and a position between the plurality of photodiodes and thefluorescence reaction vessel; wherein the photodiodes include a highconcentration first-conductivity-type semiconductor layer, asecond-conductivity-type semiconductor layer, and a low-concentrationfirst-conductivity-type semiconductor layer that are laminated in thestated order from a surface side of the photodiodes, wherein when areverse bias is applied, a part of the high-concentrationfirst-conductivity- type semiconductor layer is not depleted, and thesecond-conductivity-type semiconductor layer is depleted, and thehigh-concentration first-conductivity-type semiconductor layerconstitutes the excitation-light-entry preventing layer.
 2. The deviceaccording to claim 1, wherein the excitation-light-entry preventinglayer is a light absorbing layer that absorbs an excitation light, andis arranged between the photodiodes and the fluorescence reactionvessel.
 3. The device according to claim 1, wherein theexcitation-light-entry preventing layer is a light interference layerthat reflects an excitation light, and is arranged between thephotodiodes and the fluorescence reaction vessel.
 4. The deviceaccording to claim 1, wherein the excitation-light-entry preventinglayer is a gas layer, and is arranged between the photodiodes and thefluorescence reaction vessel, and an excitation light is set so as to beincident in a direction such that the excitation light is reflectedtotally at an interface between the gas layer and a bottom face of diefluorescence reaction vessel.
 5. The device according to claim 1,wherein a single-strand DNA is fixed on an internal bottom face of thefluorescence reaction vessel.
 6. The device according to claim 1,wherein at least one selected from an antibody and an antigen is fixedon an internal bottom face of the fluorescence reaction vessel.
 7. Afluorescence detecting method employing the fluorescence detectingdevice according to claim 1, the method comprising: causing excitationlight to enter the fluorescence reaction vessel; and detecting afluorescence generated as a result of the entry of the excitation lightby means of the photodiodes.
 8. The device according to claim 1, whereinsignal charges generated in the plurality of photodiodes arcaccumulated, and the accumulated charge is measured to detectfluorescence.